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Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

ABSTRACT: The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase.

SUBMITTER: Sun Y

PROVIDER: S-EPMC7119097 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

Sun Ying Y Wang Zidao Z Tao Jiali J Wang Yi Y Wu Andong A Yang Ziwen Z Wang Kaimei K Shi Liqiao L Chen Yu Y Guo Deyin D

Antiviral research 20140211

The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, wh ...[more]

PMID: 24530452

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Project description:The recent pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) calls the whole world into a medical emergency. For tackling Coronavirus Disease 2019 (COVID-19), researchers from around the world are swiftly working on designing and identifying inhibitors against all possible viral key protein targets. One of the attractive drug targets is guanine-N7 methyltransferase which plays the main role in capping the 5'-ends of viral genomic RNA and sub genomic RNAs, to escape the host's innate immunity. We performed homology modeling and molecular dynamic (MD) simulation, in order to understand the molecular architecture of Guanosine-P3-Adenosine-5',5'-Triphosphate (G3A) binding with C-terminal N7-MTase domain of nsp14 from SARS-CoV-2. The residue Asn388 is highly conserved in present both in N7-MTase from SARS-CoV and SARS-CoV-2 and displays a unique function in G3A binding. For an in-depth understanding of these substrate specificities, we tried to screen and identify inhibitors from the Traditional Chinese Medicine (TCM) database. The combination of several computational approaches, including screening, MM/GBSA, MD simulations, and PCA calculations, provides the screened compounds that readily interact with the G3A binding site of homology modeled N7-MTase domain. Compounds from this screening will have strong potency towards inhibiting the substrate-binding and efficiently hinder the viral 5'-end RNA capping mechanism. We strongly believe the final compounds can become COVID-19 therapeutics, with huge international support.[Formula: see text]The focus of this study is to screen for antiviral inhibitors blocking guanine-N7 methyltransferase (N7-MTase), one of the key drug targets involved in the first methylation step of the SARS-CoV-2 RNA capping mechanism. Compounds binding the substrate-binding site can interfere with enzyme catalysis and impede 5'-end cap formation, which is crucial to mimic host RNA and evade host cellular immune responses. Therefore, our study proposes the top hit compounds from the Traditional Chinese Medicine (TCM) database using a combination of several computational approaches.Communicated by Ramaswamy H. Sarma.

Project description:BackgroundIntracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11?-hydroxysteroid dehydrogenase 1 (11?-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11?-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11?-HSD1 inhibitors.ResultsThe yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11?-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11?-HSD1 inhibitory activity. As proof of concept the correlation between 11?-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11?-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11?-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11?-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times.ConclusionsOur yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11?-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11?-HSD1 inhibitors at intracellular level.

Dataset Information

Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

Publications

Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

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